U.S. patent number 6,753,008 [Application Number 10/187,112] was granted by the patent office on 2004-06-22 for dietary supplements beneficial for the liver.
This patent grant is currently assigned to Ultra Biotech Limited. Invention is credited to Ling Yuk Cheung.
United States Patent |
6,753,008 |
Cheung |
June 22, 2004 |
Dietary supplements beneficial for the liver
Abstract
Compositions comprising a plurality of yeast cells, wherein said
plurality of yeast cells are characterized by their ability to
normalize the serum level of GPT, AP and/or LDH-5 in a mammal, said
ability resulting from their having been cultured in the presence
of an alternating electric field having a specific frequency and a
specific field strength. Also included are methods of making and
using these compositions.
Inventors: |
Cheung; Ling Yuk (New
Territories, HK) |
Assignee: |
Ultra Biotech Limited (Douglas,
GB)
|
Family
ID: |
29779997 |
Appl.
No.: |
10/187,112 |
Filed: |
June 28, 2002 |
Current U.S.
Class: |
424/439; 424/400;
424/464; 424/480; 424/489; 424/800; 435/173.1; 435/173.8; 435/243;
435/254.1; 435/255.1; 435/255.2; 435/255.21 |
Current CPC
Class: |
A61P
31/12 (20180101); C12N 13/00 (20130101); A61P
1/16 (20180101); C12N 1/16 (20130101); A23L
2/52 (20130101); A23L 33/14 (20160801); Y10S
424/80 (20130101) |
Current International
Class: |
C12N
13/00 (20060101); C12N 1/16 (20060101); A61K
047/00 (); C12N 013/00 (); C12N 001/14 (); C12N
001/16 (); C12N 001/18 () |
Field of
Search: |
;424/400,439,464,489,780,800
;435/173.1,173.8,243,255.1,255.2,255.21,FOR 100/ ;435/FOR 114/
;435/254.1 |
References Cited
[Referenced By]
U.S. Patent Documents
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1110317 |
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Oct 1995 |
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CN |
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0041373 |
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Dec 1981 |
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EP |
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2222433 |
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Oct 1974 |
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FR |
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60028893 |
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Feb 1985 |
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JP |
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415983 |
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Nov 1974 |
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RU |
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1071637 |
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Feb 1984 |
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RU |
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WO 87/02705 |
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May 1987 |
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WO |
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WO 95/04814 |
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Feb 1995 |
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WO |
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WO 99/60142 |
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Nov 1999 |
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WO |
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WO 02/20431 |
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Mar 2002 |
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WO |
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WO 02/070682 |
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Sep 2002 |
|
WO |
|
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|
Primary Examiner: Page; Thurman K.
Assistant Examiner: Evans; Charesse
Attorney, Agent or Firm: Fish & Neave Haley, Jr.; James
F. Li; Z. Ying
Claims
What is claimed is:
1. A composition comprising a plurality of yeast cells, wherein
said plurality of yeast cells are characterized by their ability to
normalize the level of serum glutamate-pyruvate Transaminase (GPT),
alkaline phosphatase (AP), or lactate dehydrogenase 5 (LDH-5) in a
mammal, said ability resulting from their having been cultured in
the presence of an alternating electric field having a frequency in
the range of 18180-18240 MHZ and a field strength in the range of
100-450 mV/cm, as compared to yeast cells not having been so
cultured.
2. The composition of claim 1, wherein said frequency is in the
range of 18205 to 18227 MHZ.
3. The composition of claim 1, wherein said field strength is in
the range of 210 to 420 mV/cm.
4. The composition of claim 1, wherein said yeast cells are of the
species selected from the group consisting of Saccharomyces
cerevisiae, Saccharomyces carlsbergensis, Saccharomyces chevalieri,
Saccharomyces delbrueckii, Saccharomyces exiguous, Saccharomyces
fermentati, Saccharomyces logos, Saccharomyces mellis,
Saccharomyces oviformis, Saccharomyces rosei, Saccharomyces rouxii,
Saccharomyces sake, Saccharomyces uvarum, Saccharomyces willianus,
Saccharomyces sp., Schizosaccharomyces octosporus,
Schizosaccharomyces pombe, Sporobolomyces roseus, Torulopsis
candida, Torulopsis famta, Torulopsis globosa, Torulopsis
inconspicua, Trichosporon behrendii, Trichosporon capitatum,
Trichosporon cutaneum, Wickerhamia fluoresens, Candida arborea,
Candida krusei, Candida lambica, Candida lipolytica, Candida
parapsilosis, Candida pulcherrima, Candida rugousa, Candida
tropicalis, Candida utilis, Crebrothecium ashbyii, Geotrichum
candidum, Hansenula anomala, Hansenula arabitolgens, Hansenula
jadinii, Hansenula saturnus, Hansenula schneggii, Hansenula
subpelliculosa, Kloeckera apiculata, Lipomyces starkeyi, Pichia
farinosa, Pichia membranaefaciens, Rhodosporidium toruloides,
Rhodotorula glutinis, Rhodotorula minuta, Rhodotorula rubar,
Rhodotorula aurantiaca, Saccharomycodes ludwigii, and
Saccharomycodes sinenses.
5. The composition of claim 1, wherein said yeast cells are of the
strain deposited at the China General Microbiological Culture
Collection Center with an accession number selected from the group
consisting of Saccharomyces cerevisiae Hansen AS2.375, AS2.501,
AS2.502, AS2.503, AS2.504, AS2.535, AS2.558, AS2.560, AS2.561,
AS2.562 and IFFI1048, and Saccharomyces carlsbergensis Hansen
AS2.420 and AS2.444.
6. The composition of claim 1, wherein said composition is in the
form of a tablet, powder, or a health drink.
7. The composition of claim 1, wherein said composition is in the
form of a health drink.
8. A method of treating hepatitis in a subject, comprising
introducing orally the composition of claim 1 to the subject.
9. A method of preparing a yeast composition, comprising culturing
a plurality of yeast cells in the presence of an alternating
electric field having a frequency in the range of 18180-18240 MHZ
and a field strength in the range of 100-450 mV/cm for a period of
time sufficient to substantially increase the capability of said
plurality of yeast cells to normalize the level of serum GPT, AP,
or LDH-5 in a mammal with liver problems.
Description
FIELD OF THE INVENTION
The invention relates to compositions that are beneficial for the
liver and useful as dietary supplements. These compositions contain
yeast cells obtainable by growth in electromagnetic fields with
specific frequencies and field strengths.
BACKGROUND OF THE INVENTION
There are various types of liver diseases, including acute
hepatitis, chronic hepatitis, toxic liver injury, hepatic cancer,
cirrhotic liver, fatty liver, portal hypertension, and the like.
Liver disease in some patients develops into hepatic cirrhosis or
even hepatic cancer after a period of time (A report by the
research group on liver diseases, Health and Welfare Ministry,
1979). Prevention, observation, and cure of hepatitis are therefore
important for preventing cirrhotic liver and hepatic cancer. In
recent years, animal models of hepatitis and hepatic cancers have
been developed and their application to the research of liver
diseases is ongoing (Mori et al., Hepatic, Cholecyst, Pancresto
19(5):905-910 (1989)).
Rest and diet are principal means for curing acute hepatitis, while
various other measures are taken to cure active-type chronic
hepatitis, especially hepatitis B. Interferon, adenine arabinoside,
and acyclovir have been used to treat hepatitis. However, prolonged
use of these drugs causes severe side effects. Development of a
treatment that is safe and effective for treating liver diseases is
therefore strongly desired.
SUMMARY OF THE INVENTION
This invention is based on the discovery that certain yeast cells
can be activated by electromagnetic fields having specific
frequencies and field strengths to produce substances that are
beneficial for the liver. Compositions comprising these activated
yeast cells can be used as dietary supplements for improving liver
health, e.g., alleviating symptoms of hepatitis, cirrhosis, fatty
liver and other liver ailments.
This invention embraces a composition comprising a plurality of
yeast cells that have been cultured in an alternating electric
field having a frequency in the range of about 18000-18500 MHZ
(e.g., 18180-18240 MHz), and a field intensity in the range of
about 50 to 500 mV/cm (e.g., 100-450 mV/cm). The yeast cells are
cultured in the alternating electric field for a period of time
sufficient to substantially increase the capability of said
plurality of yeast cells to produce substances beneficial for the
liver. For instance, the cultured yeast cells when ingested can
normalize the level of serum glutamate-pyruvate Transaminase (GPT),
alkaline phosphatase (AP), and/or lactate dehydrogenase 5 (LDH-5)
in a mammal.
The term "normalize" means changing the level of abnormally high or
low concentrations of subject proteins in a mammal to a
substantially normal level.
In one embodiment, the frequency and/or the field strength of the
alternating electric field can be altered within the aforementioned
ranges during said period of time. In other words, the yeast cells
can be exposed to a series of electromagnetic fields. An exemplary
period of time is about 40-100 hours (e.g., 50 to 80 hours).
Yeast cells that can be included in this composition can all be
obtained from the China General Microbiological Culture Collection
Center ("CGMCC"), a depository recognized under the Budapest Treaty
(China Committee for Culture Collection of Microorganisms,
Institute of Microbiology, Chinese Academy of Sciences, Haidian,
P.O. BOX 2714, Beijing, 100080, China). Useful yeast species
include, but are not limited to, Saccharomyces cerevisiae,
Saccharomyces carlsbergensis, Saccharomyces chevalieri,
Saccharomyces delbrueckii, Saccharomyces exiguous, Saccharomyces
fermentati, Saccharomyces logos, Saccharomyces mellis,
Saccharomyces oviformis, Saccharomyces rosei, Saccharomyces rouxii,
Saccharomyces sake, Saccharomyces uvarum, Saccharomyces willianus,
Saccharomyces sp., Schizosaccharomyces octosporus,
Schizosaccharomyces pombe, Sporobolomyces roseus, Torulopsis
candida, Torulopsis famta, Torulopsis globosa, Torulopsis
inconspicua, Trichosporon behrendii, Trichosporon capitatum,
Trichosporon cutaneum, Wickerhamia fluoresens, Candida arborea,
Candida krusei, Candida lambica, Candida lipolytica, Candida
parapsilosis, Candida pulcherrima, Candida rugousa, Candida
tropicalis, Candida utilis, Crebrothecium ashbyii, Geotrichum
candidum, Hansenula anomala, Hansenula arabitolgens, Hansenula
jadinii, Hansenula saturnus, Hansenula schneggii, Hansenula
subpelliculosa, Kloeckera apiculata, Lipomyces starkeyi, Pichia
farinosa, Pichia membranaefaciens, Rhodosporidium toruloides,
Rhodotorula glutinis, Rhodotorula minuta, Rhodotorula rubar,
Rhodotorula aurantiaca, Saccharomycodes ludwigii, and
Saccharomycodes sinenses. For instance, the yeast cells can be of
the strain Saccharomyces cerevisiae Hansen AS2.375, AS2.501,
AS2.502, AS2.503, AS2.504, AS2.535, AS2.558, AS2.560, AS2.561,
AS2.562, or IFFI1048; or Saccharomyces carlsbergensis Hansen
AS2.420, or AS2.444.
Unless otherwise defined, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Exemplary methods and materials are described below, although
methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention. All publications and other references mentioned herein
are incorporated by reference in their entirety. In case of
conflict, the present specification, including definitions, will
control. The materials, methods, and examples are illustrative only
and not intended to be limiting. Throughout this specification and
claims, the word "comprise," or variations such as "comprises" or
"comprising" will be understood to imply the inclusion of a stated
integer or group of integers but not the exclusion of any other
integer or group of integers.
Other features and advantages of the invention will be apparent
from the following detailed description, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic diagram showing an exemplary apparatus for
activating yeast cells using electromagnetic fields. 1: yeast
culture; 2: container; 3: power supply.
FIG. 2 is a schematic diagram showing an exemplary apparatus for
making yeast compositions of the invention. The apparatus comprises
a signal generator and interconnected containers 1, 2 and 3.
DETAILED DESCRIPTION OF THE INVENTION
This invention is based on the discovery that certain yeast strains
can be activated by electromagnetic fields ("EMF") having specific
frequencies and field strengths to produce agents useful in
treating liver ailments. Yeast compositions containing the
activated yeast cells can be used as dietary supplements in the
form of health drinks or pills. In certain embodiments, the yeast
compositions of this invention can improve liver functions, thereby
normalizing the serum levels of glutamate-pyruvate transaminase,
alkaline phosphatase and/or lactate dehydrogenase 5.
Since the activated yeast cells contained in these yeast
compositions have been cultured to endure acidic conditions
(pH2.5-4.2), the compositions are stable in the stomach and can
pass on to the intestines. Once in the intestines, the yeast cells
are ruptured by various digestive enzymes, and the bioactive agents
are released and readily absorbed.
Without being bound by any theory or mechanism, the inventor
believes that EMFs activate or enhance the expression of a gene or
a set of genes or alter the conformation and/or activity of certain
cellular components (e.g. DNA, RNA, enzymes/proteins) in the yeast
cells, resulting in the production of agents that are beneficial
for the liver.
I. Yeast Strains Useful in the Invention
The types of yeasts useful in this invention include, but are not
limited to, yeasts of the genera Saccharomyces, Candida,
Crebrothecium, Geotrichum, Hansenula, Kloeckera, Lipomyces, Pichia,
Rhodosporidium, Rhodotorula, Saccharomycodes, Schizosaccharomyces,
Sporobolomyces, Torulopsis, Trichosporon, and Wickerhamia.
Exemplary species within the above-listed genera include, but are
not limited to, the species illustrated in Table 1. Yeast strains
useful in this invention can be obtained from laboratory cultures,
or from publically accessible culture depositories, such as CGMCC
and the American Type Culture Collection, 10801 University
Boulevard, Manassas, Va. 20110-2209. Non-limiting examples of
useful strains (with the accession numbers of CGMCC) are
Saccharomyces cerevisiae Hansen AS2.375, AS2.501, AS2.502, AS2.503,
AS2.504, AS2.535, AS2.558, AS2.560, AS2.561, AS2.562, and IFFI1048;
and Saccharomyces carlsbergensis Hansen AS2.420 and AS2.444. Other
non-limiting examples of useful strains are listed in Table 1. In
general, yeast strains preferred in this invention are those used
for fermentation in the food and wine industries. As a result,
compositions containing these yeast cells are safe for human
consumption.
Although it is preferred, the preparation of the yeast compositions
of this invention is not limited to starting with a pure strain of
yeast. A yeast composition of the invention may be produced by
culturing a mixture of yeast cells of different species or
strains.
TABLE 1 Exemplary Yeast Strains Saccharomyces cerevisiae Hansen
ACCC2034 ACCC2035 ACCC2036 ACCC2037 ACCC2038 ACCC2039 ACCC2040
ACCC2041 ACCC2042 AS2. 1 AS2. 4 AS2. 11 AS2. 14 AS2. 16 AS2. 56
AS2. 69 AS2. 70 AS2. 93 AS2. 98 AS2. 101 AS2. 109 AS2. 110 AS2. 112
AS2. 139 AS2. 173 AS2. 174 AS2. 182 AS2. 196 AS2. 242 AS2. 336 AS2.
346 AS2. 369 AS2. 374 AS2. 375 AS2. 379 AS2. 380 AS2. 382 AS2. 390
AS2. 393 AS2. 395 AS2. 396 AS2. 397 AS2. 398 AS2. 399 AS2. 400 AS2.
406 AS2. 408 AS2. 409 AS2. 413 AS2. 414 AS2. 415 AS2. 416 AS2. 422
AS2. 423 AS2. 430 AS2. 431 AS2. 432 AS2. 451 AS2. 452 AS2. 453 AS2.
458 AS2. 460 AS2. 463 AS2. 467 AS2. 486 AS2. 501 AS2. 502 AS2. 503
AS2. 504 AS2. 516 AS2. 535 AS2. 536 AS2. 558 AS2. 560 AS2. 561 AS2.
562 AS2. 576 AS2. 593 AS2. 594 AS2. 614 AS2. 620 AS2. 628 AS2. 631
AS2. 666 AS2. 982 AS2. 1190 AS2. 1364 AS2. 1396 IFFI1001 IFFI1002
IFFI1005 IFFI1006 IFFI1008 IFFI1009 IFFI1010 IFFI1012 IFFI1021
IFFI1027 IFFI1037 IFFI1042 IFFI1043 IFFI1045 IFFI1048 IFFI1049
IFFI1050 IFFI1052 IFFI1059 IFFI1060 IFFI1062 IFFI1063 IFFI1202
IFFI1203 IFFI1206 IFFI1209 IFFI1210 IFFI12II IFFI1212 IFFI1213
IFFI1214 IFFI1215 IFFI1220 IFFI1221 IFFI1224 IFFI1247 IFFI1248
IFFI1251 IFFI1270 IFFI1277 IFFI1287 IFFI1289 IFFI1290 IFFI1291
IFFI1292 IFFI1293 IFFI1297 IFFI1300 IFFI1301 IFFI1302 IFFI1307
IFFI1308 IFFI1309 IFFI1310 IFFI1311 IFFI1331 IFFI1335 IFFI1336
1FFI1337 IFFI1338 IFFI1339 IFFI1340 IFFI1345 IFFI1348 IFFI1396
IFFI1397 1FFI1399 IFFI1411 IFFI1413 IFFI1441 IFFI1443 Saccharomyces
cerevisiae Hansen Var. ellipsoideus (Hansen) Dekker ACCC2043 AS2.2
AS2.3 AS2.8 AS2.53 AS2.163 AS2.168 AS2.483 AS2.541 AS2.559 AS2.606
AS2.607 AS2.611 AS2.612 Saccharomyces chevalieri Guilliermond
AS2.131 AS2.213 Saccharomyces delbrueckii AS2.285 Saccharomyces
delbrueckii Lindner ver. mongolicus (Saito) Lodder et van Rij
AS2.209 AS2.1157 Saccharomyces exiguous Hansen AS2.349 AS2.1158
Saccharomyces fermentati (Saito) Lodder et van Rij AS2.286 AS2.343
Saccharomyces logos van laer et Denamur ex Jorgensen AS2.156
AS2.327 AS2.335 Saccharomyces mellis (Fabian et Quinet) Lodder et
kreger van Rij AS2.195 Saccharomyces mellis Microellipsoides
Osterwalder AS2.699 Saccharomyces oviformis Osteralder AS2.100
Saccharomyces rosei (Guilliermond) Lodder et Kreger van Rij AS2.287
Saccharomyces rouxii Boutroux AS2.178 AS2.180 AS2.370 AS2.371
Saccharomyces sake Yabe ACCC2045 Candida arborea AS2.566 Candida
lambica (Lindner et Genoud) van. Uden et Buckley AS2.1182 Candida
krusei (Castellani) Berkhout AS2.1045 Candida lipolytica (Harrison)
Diddens et Lodder AS2.1207 AS2.1216 AS2.1220 AS2.1379 AS2.1398
AS2.1399 AS2.1400 Candida parapsilosis (Ashford) Langeron et Talice
Var. intermedia Van Rij et Verona AS2.491 Candida parapsilosis
(Ashford) Langeron et Talice AS2.590 Candida pulcherrima (Lindner)
Windisch AS2.492 Candida rugousa (Anderson) Diddens et Lodder
AS2.511 AS2.1367 AS2.1369 AS2.1372 AS2.1373 AS2.1377 AS2.1378
AS2.1384 Candida tropicalis (Castellani) Berkhout ACCC2004 ACCC2005
ACCC2006 AS2.164 AS2.402 AS2.564 AS2.565 AS2.567 AS2.568 AS2.617
AS2.637 AS2.1387 AS2.1397 Candida utilis Henneberg Lodder et Kreger
Van Rij AS2.120 AS2.281 AS2.1180 Crebrothecium ashbyii
(Guillermond) Routein (Eremothecium ashbyii Guilliermond) AS2.481
AS2.482 AS2.1197 Geotrichum candidum Link ACCC2016 AS2.361 AS2.498
AS2.616 AS2.1035 AS2.1062 AS2.1080 AS2.1132 AS2.1175 AS2.1183
Hansenula anomala (Hansen)H et P sydow ACCC2018 AS2.294 AS2.295
AS2.296 AS2.297 AS2.298 AS2.299 AS2.300 AS2.302 AS2.338 AS2.339
AS2.340 AS2.341 AS2.470 AS2.592 AS2.641 AS2.642 AS2.782 AS2.635
AS2.794 Hansenula arabitolgens Fang AS2.887 Hansenula jadinii (A.
et R Sartory Weill et Meyer) Wickerham ACCC2019 Hansenula saturnus
(Klocker) H et P sydow ACCC2020 Hansenula schneggii (Weber) Dekker
AS2.304 Hansenula subpelliculosa Bedford AS2.740 AS2.760 AS2.761
AS2.770 AS2.783 AS2.790 AS2.798 AS2.866 Kloeckera apiculata (Reess
emend. Klocker) Janke ACCC2022 ACCC2023 AS2.197 AS2.496 AS2.714
ACCC2021 AS2.711 Lipomycess starkeyi Lodder et van Rij AS2.1390
ACCC2024 Pichia farinosa (Lindner) Hansen ACCC2025 ACCC2026 AS2.86
AS2.87 AS2.705 AS2.803 Pichia membranaefaciens Hansen ACCC2027
AS2.89 AS2.661 AS2.1039 Rhodosporidium toruloides Banno ACCC2028
Rhodotorula glutinis (Fresenius) Harrison AS2.2029 AS2.280 ACCC2030
AS2.102 AS2.107 AS2.278 AS2.499 AS2.694 AS2.703 AS2.704 AS2.1146
Rhodotorula minuta (Saito) Harrison AS2.277 Rhodotorula rubar
(Demme) Lodder AS2.21 AS2.22 AS2.103 AS2.105 AS2.108 AS2.140
AS2.166 AS2.167 AS2.272 AS2.279 AS2.282 ACCC2031 Rhodotorula
aurantiaca (Saito) Lodder AS2.102 AS2.107 AS2.278 AS2.499 AS2.694
AS2.703 AS2.704 AS2.1146 Saccharomyces carlsbergensis Hansen
AS2.113 ACCC2032 ACCC2033 AS2.312 AS2.116 AS2.118 AS2.121 AS2.132
AS2.162 AS2.189 AS2.200 AS2.216 AS2.265 AS2.377 AS2.417 AS2.420
AS2.440 AS2.441 AS2.443 AS2.444 AS2.459 AS2.595 AS2.605 AS2.638
AS2.742 AS2.745 AS2.748 AS2.1042 Saccharomyces uvarum Beijer
IFF11023 IFFI1032 IFFI1036 1FFI1044 IFFI1072 IFFI1205 IFFI1207
Saccharomyces willianus Saccardo AS2.5 AS2.7 AS2.119 AS2.152
AS2.293 AS2.381 AS2.392 AS2.434 AS2.614 AS2.1189 Saccharomyces sp.
AS2.31 1 Saccharomycodes ludwigii Hansen ACCC2044 AS2.243 AS2.508
Saccharomycodes sinenses Yue AS2.1395 Schizosaccharomyces
octosporus Beijerinck ACCC2046 AS2.1148 Schizosaccharomyces pombe
Lindner ACCC2047 ACCC2048 AS2.214 AS2.248 AS2.249 AS2.255 AS2.257
AS2.259 AS2.260 AS2.274 AS2.994 AS2.1043 AS2.1149 AS2.1178 IFFI1056
Sporobolomyces roseus Kluyver et van Niel ACCC2049 ACCC2050 AS2.19
AS2.962 AS2.1036 ACCC2051 AS2.261 AS2.262 Torulopsis candida
(Saito) Lodder AS2.270 ACCC2052 Torulopsis famta (Harrison) Lodder
et van Rij ACCC2053 AS2.685 Torulopsis globosa (Olson et Hammer)
Lodder et van Rij ACCC2054 AS2.202 Torulopsis inconspicua Lodder et
Kreger van Rij AS2.75 Trichosporon behrendii Lodder et. Kreger van
Rij ACCC2056 AS2.1193 Trichosporon capitatum Diddens et Lodder
ACCC2056 AS2.1385 Trichosporon cutaneum (de Beurm et al.) Ota
ACCC2057 AS2.25 AS2.570 AS2.571 AS2.1374 Wickerhamia fluorescens
(Soneda) Soneda ACCC2058 AS2.1388
II. Application of Electromagnetic Fields
An electromagnetic field useful in this invention can be generated
and applied by various means well known in the art. For instance,
the EMF can be generated by applying an alternating electric field
or an oscillating magnetic field.
Alternating electric fields can be applied to cell cultures through
electrodes in direct contact with the culture medium, or through
electromagnetic induction. See, e.g., FIG. 1. Relatively high
electric fields in the medium can be generated using a method in
which the electrodes are in contact with the medium. Care must be
taken to prevent electrolysis at the electrodes from introducing
undesired ions into the culture and to prevent contact resistance,
bubbles, or other features of electrolysis from dropping the field
level below that intended. Electrodes should be matched to their
environment, for example, using Ag-AgCl electrodes in solutions
rich in chloride ions, and run at as low a voltage as possible. For
general review, see Goodman et al., Effects of EMF on Molecules and
Cells, International Review of Cytology, A Survey of Cell Biology,
Vol. 158, Academic Press, 1995.
The EMFs useful in this invention can also be generated by applying
an oscillating magnetic field. An oscillating magnetic field can be
generated by oscillating electric currents going through Helmholtz
coils. Such a magnetic field in turn induces an electric field.
The frequencies of EMFs useful in this invention range from about
18000 MHZ to 18500 MHZ. Exemplary frequencies include 18205, 18211,
18217, 18223, and 18227 MHZ. The field strength of the electric
field useful in this invention ranges from about 100 to 450 mV/cm
(e.g., 100-150, 210-260, 300-340, or 380-420 mV/cm). Exemplary
field strengths include 240, 248, 408, 415, and 315 mV/cm.
When a series of EMFs are applied to a yeast culture, the yeast
culture can remain in the same container while the same set of EMF
generator and emitters is used to change the frequency and/or field
strength. The EMFs in the series can each have a different
frequency or a different field strength; or a different frequency
and a different field strength. Such frequencies and field
strengths are preferably within the above-described ranges.
Although any practical number of EMFs can be used in a series, it
may be preferred that the yeast culture be exposed to a total of 2,
3, 4, 5, 6, 7, 8, 9 or 10 EMFs in a series.
Although the yeast cells can be activated after even a few hours of
culturing in the presence of an EMF, it may be preferred that the
activated yeast cells be allowed to multiply and grow in the
presence of the EMF(s) for a total of 40-100 hours.
FIG. 1 illustrates an exemplary apparatus for generating
alternating electric fields. An electric field of a desired
frequency and intensity can be generated by an AC source (3)
capable of generating an alternating electric field, preferably in
a sinusoidal wave form, in the frequency range of 5 to 20,000 MHZ.
Signal generators capable of generating signals with a narrower
frequency range can also be used. If desired, a signal amplifier
can also be used to increase the output. The culture container (2)
can be made from a non-conductive material, e.g., glass, plastic or
ceramic. The cable connecting the culture container (2) and the
signal generator (3) is preferably a high frequency coaxial cable
with a transmission frequency of at least 30 GHz.
The alternating electric field can be applied to the culture by a
variety of means, including placing the yeast culture (1) in close
proximity to the signal emitters such as a metal wire or tube
capable of transmitting EMFs. The metal wire or tube can be made of
red copper, and be placed inside the container (2), reaching as
deep as 3-30 cm. For example, if the fluid in the container (2) has
a depth of 15-20 cm, 20-30 cm, 30-50 cm, 50-70 cm, 70-100 cm,
100-150 cm or 150-200 cm, the metal wire can be 3-5 cm, 5-7 cm,
7-10 cm, 10-15 cm, 15-20 cm, 20-30 cm and 25-30 cm from the bottom
of the container (2), respectively. The number of metal wires/tubes
used can be from 1 to 10 (e.g., 2 to 3). It is recommended, though
not mandated, that for a culture having a volume up to 10 L, metal
wires/tubes having a diameter of 0.5 to 2 mm be used. For a culture
having a volume of 10-100 L, metal wires/tubes having a diameter of
3 to 5 mm can be used. For a culture having a volume of 100-1000 L,
metal wires/tubes having a diameter of 6 to 15 mm can be used. For
a culture having a volume greater than 1000 L, metal wires/tubes
having a diameter of 20-25 mm can be used.
In one embodiment, the electric field is applied by electrodes
submerged in the culture (1). In this embodiment, one of the
electrodes can be a metal plate placed on the bottom of the
container (2), and the other electrode can comprise a plurality of
electrode wires evenly distributed in the culture (1) so as to
achieve even distribution of the electric field energy. The number
of electrode wires used depends on the volume of the culture as
well as the diameter of the wires.
III. Culture Media
Culture media useful in this invention contain sources of nutrients
that can be assimilated by yeast cells. Complex carbon-containing
substances in a suitable form (e.g., carbohydrates such as sucrose,
glucose, dextrose, maltose and xylose) can be the carbon sources
for yeast cells. The exact quantity of the carbon sources can be
adjusted in accordance with the other ingredients of the medium. In
general, the amount of carbohydrate varies between about 0.1% and
10% by weight of the medium and preferably between about 0.1% and
5%, and most preferably about 2%. These carbon sources can be used
individually or in combination. Amino acid-containing substances
such as beef extract and peptone can also be added. In general, the
amount of amino acid containing substances varies between about
0.1% and 1% by weight of the medium and preferably between about
0.1% and 0.5%. Among the inorganic salts which can be added to a
culture medium are the customary salts capable of yielding sodium,
potassium, calcium, phosphate, sulfate, carbonate, and like ions.
Non-limiting examples of nutrient inorganic salts are
(NH.sub.4).sub.2 HPO.sub.4, CaCO.sub.3, KH.sub.2 PO.sub.4, K.sub.2
HPO.sub.4, MgSO.sub.4, NaCl, and CaSO.sub.4.
IV. Electromagnetic Activation of Yeast Cells
To activate or enhance the ability of yeast cells to produce agents
beneficial for the gastrointestinal system, these cells can be
cultured in an appropriate medium under sterile conditions at
20-35.degree. C. (e.g., 28-32.degree. C.) for a sufficient amount
of time (e.g., 10-150 hours) in an alternating electric field or a
series of alternating electric fields as described above.
An exemplary set-up of the culture process is depicted in FIG. 1
(see above). An exemplary culture medium contains the following per
1000 ml of sterile water: 20 g of sucrose, 40 .mu.g of Vitamin B1,
50 .mu.g of Vitamin B6, 0.2 g of KH.sub.2 PO.sub.4, 0.2 g of
MgSO.sub.4.7H.sub.2 O, 0.25 g of NaCl, 0.1 g of CaSO.sub.4.2H.sub.2
O, 3 g of CaCO.sub.3.5H.sub.2 O, and 2.5 g of peptone. Yeast cells
of the desired strain(s) are then added to the culture medium to
form a mixture containing 1.times.10.sup.8 cells per 1000 ml of
culture medium. The yeast cells can be of any of the strains listed
in Table 1. The mixture is then added to the apparatus shown in
FIG. 1.
The activation process of the yeast cells involves the following
steps: (1) maintaining the temperature of the activation apparatus
at 24-33.degree. C. (e.g., 28-32.degree. C.), and culturing the
yeast cells for 30-42 hours (e.g., 38 hours); (2) applying an
alternating electric field having a frequency of 18205 MHZ and a
field strength of 210-260 mV/cm (e.g., 239-241 mV/cm) for 13-20
hours (e.g., 15 hours); (3) then applying an alternating electric
field having a frequency of 18211 MHZ and a field strength of
220-260 mV/cm (e.g., 247-249 mV/cm) for 15-25 hours (e.g., 19
hours); (4) then applying an alternating electric field having a
frequency of 18217 MHZ and a field strength of 380-420 mV/cm (e.g.,
406-410 mV/cm) for 20-30 hours (e.g., 25 hours); (5) then applying
an alternating electric field having a frequency of 18223 MHZ and a
field strength of 380-420 mV/cm (e.g., 413-417 mV/cm) for 9-12
hours (e.g., 10 hours); and (6) then applying an alternating
electric field having a frequency of 18227 MHZ and a field strength
of 300-330 mV/cm (e.g., 313-317 mV/cm) for 9-12 hours (e.g., 10
hours). The activated yeast cells are then recovered from the
culture medium by various methods known in the art, dried (e.g., by
lyophilization) and stored at 4.degree. C. Preferably, the
concentration of the dried yeast cells are no less than 10.sup.10
cells/g.
V. Acclimatization of Yeast Cells to the Gastric Environment
Because the yeast compositions of this invention must pass through
the stomach before reaching the small intestine, where the
effective components are released from these yeast cells, it is
preferred that these yeast cells be cultured under acidic
conditions to acclimatize the cells to the gastric juice. This
acclimatization process results in better viability of the yeast
cells in the acidic gastric environment.
To achieve this, the yeast powder containing activated yeast cells
can be mixed with a highly acidic acclimatizing culture medium at
10 g (containing more than 10.sup.10 activated cells per gram) per
1000 ml. The yeast mixture is then cultured first in the presence
of an alternating electric field having a frequency of 18223 MHZ
and a field strength of 390-420 mV/cm (e.g., 403-407 mV/cm) at
about 28 to 32.degree. C. for 25 to 48 hours (e.g., 46 hours). The
resultant yeast cells can then be further incubated in the presence
of an alternating electric field having a frequency of 18227 MHZ
and a field strength of 300-330 mV/cm (e.g., 315-319 mV/cm) at
about 28 to 32.degree. C. for 15 to 25 hours (e.g., 20 hours). The
resulting acclimatized yeast cells are then either dried and stored
in powder form (.gtoreq.10.sup.10 cells/g) at room temperature or
in vacuum at 0-4.degree. C.
An exemplary acclimatizing culture medium is made by mixing 700 ml
fresh pig gastric juice and 300 ml wild Chinese hawthorn extract.
The pH of the acclimatizing culture medium is adjusted to 2.5 with
0.1 M hydrochloric acid (HCl) and/or 0.2 M potassium biphthalate
(C.sub.6 H.sub.4 (COOK)COOH). The fresh pig gastric juice is
prepared as follows. At about 4 months of age, newborn Holland
white pigs are sacrificed, and the entire contents of their
stomachs are retrieved and mixed with 2000 ml of water under
sterile conditions. The mixture is then allowed to stand for 6
hours at 4.degree. C. under sterile conditions to precipitate food
debris. The supernatant is collected for use in the acclimatizing
culture medium. To prepare the wild Chinese hawthorn extract, 500 g
of fresh wild Chinese hawthorn is dried under sterile conditions to
reduce water content (.ltoreq.8%). The dried fruit is then ground
(.gtoreq.20 mesh) and added to 1500 ml of sterile water. The
hawthorn slurry is allowed to stand for 6 hours at 4.degree. C.
under sterile conditions. The hawthorn supernatant is collected to
be used in the acclimatizing culture medium.
VI. Manufacture of Yeast Compositions
To manufacture the yeast compositions of the invention, an
apparatus depicted in FIG. 2 or an equivalent thereof can be used.
This apparatus includes three containers, a first container (1), a
second container (2), and a third container (3), each equipped with
a pair of electrodes (4). One of the electrodes is a metal plate
placed on the bottom of the containers, and the other electrode
comprises a plurality of electrode wires evenly distributed in the
space within the container to achieve even distribution of the
electric field energy. All three pairs of electrodes are connected
to a common signal generator.
The culture medium used for this purpose is a mixed fruit extract
solution containing the following ingredients per 1000 L: 300 L of
wild Chinese hawthorn extract, 300 L ofjujube extract, 300 L of Wu
Wei Zi (Schisandra chinensis (Turez) Baill seeds) extract, and 100
L of soy bean extract. To prepare hawthorn, jujube and Wu Wei Zi
extracts, the fresh fruits are washed and dried under sterile
conditions to reduce the water content to no higher than 8%. One
hundred kilograms of the dried fruits are then ground (.gtoreq.20
mesh) and added to 400 L of sterile water. The mixtures are stirred
under sterile conditions at room temperature for twelve hours, and
then centrifuged at 1000 rpm to remove insoluble residues. To make
the soy bean extract, fresh soy beans are washed and dried under
sterile conditions to reduce the water content to no higher than
8%. Thirty kilograms of dried soy beans are then ground into
particles of no smaller than 20 mesh, and added to 130 L of sterile
water. The mixture is stirred under sterile conditions at room
temperature for twelve hours and then centrifuged at 1000 rpm to
remove insoluble residues. To make the culture medium, these
extracts are mixed according to the above recipe, and the mixture
is autoclaved at 121.degree. C. for 30 minutes and cooled to below
40.degree. C. before use.
One thousand grams of the activated yeast powder prepared as
described above (Section V, supra) is added to 1000 L of the mixed
fruit extract solution, and the yeast solution is transferred to
the first container (1) shown in FIG. 2. The yeast cells are then
cultured in the presence of an altemating electric field having a
frequency of 18223 MHZ and a field strength of about 390-420 mV/cm
(e.g., 403-407 mV/cm) at 28-32.degree. C. under sterile conditions
for 16 hours. The yeast cells are further incubated in an
alternating electric field having a frequency of 18227 MHZ and a
field strength of 320-350 mV/cm (e.g., 333-337 mV/cm). The
culturing continues for another 12 hours.
The yeast culture is then transferred from the first container (1)
to the second container (2) which contains 1000 L of culture medium
(if need be, a new batch of yeast culture can be started in the now
available first container (1)), and subjected to an alternating
electric field having a frequency of 18223 MHZ and a field strength
of 200-220 mV/cm (e.g., 206-210 mV/cm) for 10 hours. Subsequently
the frequency and field strength of the electric field are changed
to 18227 MHZ and 210-230 mV/cm (e.g., 213-217 mV/cm), respectively.
The culturing continues for another ten hours.
The yeast culture is then transferred from the second container (2)
to the third container (3) which contains 1000 L of culture medium,
and subjected to an alternating electric field having a frequency
of 18223 MHZ and a field strength of 90-110 mV/cm (e.g., 104-108
mV/cm) for 12 hours. Subsequently the frequency and field strength
of the electric field are changed to 18227 MHZ and 100-120 mV/cm
(e.g., 103-107 mV/cm), respectively. The culturing continues for
another 8 hours.
The yeast culture from the third container (3) can then be packaged
into vacuum sealed bottles for use as dietary supplement, e.g.,
health drinks. If desired, the final yeast culture can also be
dried within 24 hours and stored in powder form. The dietary
supplement can be taken three to four times daily at 30-60 ml/dose
for a three-month period, preferably 10-30 minutes before meals and
at bedtime.
In some embodiments, the compositions of the invention can also be
administered intravenously or peritoneally in the form of a sterile
injectable preparation. Such a sterile preparation can be prepared
as follows. A sterilized health drink composition is first treated
under ultrasound (1000 Hz) for 10 minutes and then centrifuged at
4355 g for another 10 minutes. The resulting supernatant is
adjusted to pH 7.2-7.4 using 1 M NaOH and subsequently filtered
through a membrane (0.22 .mu.m for intravenous injection and 0.45
.mu.m for peritoneal injection) under sterile conditions. The
resulting sterile preparation is submerged in a 35-38.degree. C.
water bath for 30 minutes before use.
The yeast compositions of the present invention are derived from
yeasts used in food and pharmaceutical industries. The yeast
compositions are thus devoid of side effects associated with many
pharmaceutical compounds
VII. EXAMPLES
The following examples are meant to illustrate the methods and
materials of the present invention. Suitable modifications and
adaptations of the described conditions and parameters which are
obvious to those skilled in the art are within the spirit and scope
of the present invention.
The activated yeast compositions used in the following experiments
were prepared as described above, using Saccharomyces cerevisiae
Hansen AS2.560 cells cultured in the presence of an alternating
electric field having the electric field frequency and field
strength exemplified in the parentheses following the recommended
ranges listed in Section IV, supra. Control yeast compositions were
those prepared in the same manner except that the yeast cells were
cultured in the absence of EMFs. Unless otherwise indicated, the
yeast compositions and the corresponding controls were administered
to the animals by intragastric feeding.
Example 1
Serum Glutamate-pyruvate Transaminase Activity
Glutamate-pyruvate transaminase (GPT) normally is expressed in
hepatocytes. When the liver tissue undergoes necrosis or is
otherwise damaged, GPT is released into the blood stream, elevating
the level of serum GPT. Thus, the serum GPT level is one of the
important indicators of liver functions.
In this study, 32 Wistar rats (170-200 g, 8-10 months old) were
divided into 4 groups, each having 4 females and 4 males. Rats in
group A were each given 3 ml of the activated yeast composition
once daily for 8 days. On days 1 and 5, the rats were also injected
with 5 mg of carbon tetrachloride per kilogram body weight. Rats in
groups B and C were treated in the same manner except that the rats
were given the control yeast composition and saline, respectively,
in lieu of the activated yeast composition. Rats in group D were
treated in the same manner as group C except that no carbon
tetrachloride was administered. On day 8, the rats were sacrificed,
and their blood was drawn to determine serum GPT levels.
To do so, 0.1 ml of serum from each animal was mixed with 0.5 ml of
the glutamate-pyruvate substrate solution (1 M) and incubated in a
37.degree. C. water bath for 30 minutes. Then 0.5 ml of
2,4-dinitrophenylhydrazine was added and the incubation continued
for another 20 minutes. Finally 5 ml of 0.4 M NaOH was added. The
control reaction was prepared in the same manner except that the
serum was added immediately after, not before, the 30 minute
incubation step. The optical density of the sample was measured at
520 nm, using the control reaction for calibration. The GPT
concentration was determined by using a standard curve. The data
are shown in Table 2 below.
TABLE 2 Group Number of animals Serum GTP A 8 61.3 .+-. 18.64 B 8
279.6 .+-. 132.38 C 8 288.5 .+-. 126.83 D 8 101.6 .+-. 32.07
The data demonstrate that the activated yeast composition
significantly restored serum GPT to normal levels in rats treated
with carbon tetrachloride.
Example 2
Activity of Serum Alkaline Phosphatase
Serum alkaline phosphatase (AP) is produced mainly by the liver.
The level of serum AP is an indicator of the liver health, with an
elevated level suggesting an unhealthy liver.
In this study, 32 male Sprague-Dawley rats (120-150 g) were divided
into 4 equal groups. Rats in group A were each given 3 ml of the
activated yeast composition daily for 13 days. Every three days
during this time period, the animals were also injected with 2 mg
of liquid paraffin containing 15% carbon tetrachloride per kg body
weight (four times total). Rats in groups B and C were treated in
the same manner, except that they were given the control yeast
composition and saline, respectively, in lieu of the activated
yeast composition. Rats in group D were treated in the same manner
as group C except that no paraffin injection was made.
On day 13, after the last CCl.sub.4 injection, the animals were
fasted for 16 hours. Then the animals were sacrificed, and their
serum GPT and AP levels determined. GPT levels were determined as
described above. To determine AP levels, 0.1 ml of serum from the
animal was mixed with 4 ml of the AP substrate solution and
incubated in a 37.degree. C. water bath for 7 minutes. Then 1 ml of
0.6% 4-AAP (alanine aminopeptidase) and 1 ml of 4.8% K.sub.3
Fe(CN).sub.6 were added. The standard was prepared in the same
manner except that PHEN standard solution was used in lieu of
serum. For blank control, no serum or PHEN solution was added.
The optical density of the sample was then measured at 500 nm,
using the blank control to calibrate the spectrophotometer.
Alkaline phosphatase (AP) activity was calculated as [(OD of test
sample)/(OD of standard)].times.10. The experimental data are shown
in Table 3 below.
TABLE 3 Serum GPT AP (units/ Liver weight Number of (units/ml 100
ml (g/100 g Group Animals serum) serum) body weight A 8 42.2 .+-.
19.4 33.6 .+-. 5.2 3.81 .+-. 0.21 B 8 162.9 .+-. 78.3 55.9 .+-. 7.2
5.07 .+-. 0.19 C 8 167.4 .+-. 89.5 57.4 .+-. 5.5 5.19 .+-. 0.25 D 8
27.3 .+-. 7.3 42.2 .+-. 12.0 3.72 .+-. 0.30
These results indicate that, unlike the control yeast composition,
the activated yeast composition of this invention normalized serum
GPT and AP levels in rats injected with liver-damaging agents.
Example 3
Activity of Lactate Dehydrogenase 5
An elevated level of lactate dehydrogenase 5 (LDH-5) often
accompanies hepatitis caused by hepatitis B virus. In this
experiment, the effectiveness of the activated yeast composition in
treating hepatitis in a mouse model was assessed.
Liver extract prepared from hybrid mice was used to immunize
pure-bred mice to induce chronic hepatitis. Specifically, livers
from hybrid mice were minced and centrifuged in a refrigerated
centrifuge at 10,000 g for 30 minutes. The supernatant was
collected and mixed with Freund's complete adjuvant to form an
emulsion for injection into newly weaned male C57BL mice.
Forty newly weaned C57BL mice were divided into four equal groups.
Mice in group A were each administered 1 ml of the activated yeast
composition daily for 9 weeks. During the first five weeks, the
mice were each injected with 0.1 ml of the liver extract emulsion
twice weekly. During the remaining four weeks, the injection was
administered once weekly. Mice in groups B and C were treated in
the same manner, except that the control yeast composition and
saline, respectively, were used in lieu of the activated yeast
composition. Mice in group D were treated in the same manner as
group C, except that no injection of liver emulsion was
administered.
Twenty-four hours after the last day of treatment, the animals were
sacrificed and their blood sera were collected. LDH-5 was isolated
from the serum using cellulose acetate electrophoresis. And the
CICs (circulating immune complexes) were measured by PEG
(polyethylene glycol) precipitation method. The results are shown
in Table 4 below.
TABLE 4 Number of Group Animals LDH-5 (ug/L) CLC OD A 10 61 .+-.
5.21 0.025 .+-. 0.003 B 10 94 .+-. 7.31 0.059 .+-. 0.007 C 10 93
.+-. 6.41 0.061 .+-. 0.007 D 10 58 .+-. 2.47 0.023 .+-. 0.002
These data demonstrate that the activated yeast composition arkedly
reduced serum LDH-5 and CLC levels in mice with induced hepatitis,
as compared to control.
While a number of embodiments of this invention have been set
forth, it is apparent that the basic constructions may be altered
to provide other embodiments which utilize the compositions and
methods of this invention.
* * * * *